Process for preparing 1-dehydro steroids



United States Patent .0

3,022,226 PROCESS FOR PREPARING l-DEHYDRO STEROIDS John W. Ross, EastBrunswick, NJ., assignor to Olin Mathieson Chemical Corporation, NewYork, N.Y., a corporation of Virginia N Drawing. Filed Nov. 23, 1960,Ser. No. 71,140

6 Claims. (Cl. 195-51) This invention relates to a process for preparingsteroids and, more particularly, to an improved process for themicrobial l-dehydrogenation of steroids.

With the discovery that the introduction of a double bond into the1,2-position of hydrocortisone increased the glucocorticoid activity,attention was directed to processes for l-dehydrogenation of steroids ofthe 3,20-diketo- A -pregnene series. Subsequently other l-dehydrogenatedsteroids were found to have'commercial utility as glucocorticoid andanti-inflammatory drugs. Among such steroids can be mentionedtriamcinolone and dexamethasone. It was-also found that the desiredl-dehydrogenation could be accomplished either chemically ormicrobiologically. Chemical methods, such as by the use of seleniumdioxide, however, suffered the disadvantage of giving a seleniumcontaining by-product which was dificult to remove. Microbial methods,although superior, have hithertofor been accompanied by substantialreduction of the keto group in the 20-position to yield an undesiredZO-hydroxy derivative.

It is an object of this invention, therefore, to provide an improvedprocess for l-dehydrogenating a steroid of the 3,20-diketo-A -pregneneseries. i

It is another object of this invention to provide a process forl-dehydrogenating a steroid of the 3,20- diketo-n -pregnene serieswherein the concomitant reduction of the ZO-keto group to a ZO-hydroxygroup is minimized or eliminated.

These objects are achieved by the process of this invention whichcomprises subjecting under aerobic conditions a steroid of the3,2O-diketo-A -pregnene series to the action of a l-dehydrogenase enzymein the presence of an iodoacetate compound. For this purpose there isused iodoacetic acid itself; a salt thereof, such as an alkali metalsalt (e.g., sodium iodoacetate and potassium iodoacetate), an alkalineearth metal salt, the ammonium salt, and an amine salt; or an esterthereof, such as a lower alkyl ester (e.g., methyl iodoacetate and ethyliodoacetate) and a monocyclic aralkyl ester.

The l-dehydrogenase enzyme utilized in the process of this invention ispreferably prepared separately 'in an initial step wherein amicroorganism known to effect l-dehydrogenation of steroids is grown ina suitable aqueous nutrient medium containing a substance which inducesthe formation of the desired l-dehydrogenase enzyme. Suitable inducingsubstances include steroids saturated in the 1,2-position. Although anysuch steroid may be used, because of their low cost testosterone andprogesterone are particularly preferred for this purpose.

Suitable microorganisms include those known to effect .l-dehydrogenationof steroids as exemplified by members of the genera: Corynebacterium(e.g., C. simplex), Nocardia (e.g., N. aurantia and N. asteroides),Bacterium (e.g., B. cyclooxydans), Mycobacterium (e.g., M. rhodochrous),Bacillus (e.g., B. sphaericus), Septomyxa (e.g., S. afiinis), Didymella(e.g., D. Iycopersici), Calonectria (e.g., C. decora), Fusarium (e.g.,F. solzmi), Cylindro carpon (e.g., C. radicicola), Pseudomonas (e.g., P.testosteroni), Streptomyces (e.g., S. lavena'alae), and also selectedspecies of the genera Protaminobacter, Alcaligenes, Alternaria,Ophio'oolus and Pyinodithis.

In general, the conditions of culturing the microorganisms for thepurpose of preparing the l-dehydro- 3,@ZZ,ZZ6 Patented Feb. 20, 1962genase is the same as those of culturing microorganisms for theproduction of antibiotics or vitamins. Thus, the microorganism is grownin contact with (in or on) a suitable nutrient medium. if an aerobicmicroorganism is being grown, an adequate supply of oxygen (air) isprovided during the growth period. A suitable nutrient mediumessentially comprises a source of nitrogenous factors and an assimilablesource of carbon and energy. The latter may be a carbohydrate, such assucrose, molasses, glucose, maltose, starch or dextrin. The source ofnitrogenous factors may be organic (e.g., soybean meal, corn steepliquor, meat extract, distillers solubles, peptones and/or yeastextract) or synthetic (i.e., composed ofsimple, syuthesizable organicand inorganic compounds such as ammonium salts, alkali nitrates, aminoacids or urea).

In order to induce the formation of the desired 1- dehydrogenase enzyme,a 1,2-saturated steroid, such as progesterone and testosterone, is alsoaddedto the nutrientmedium. The steroid is present in suilicientquantity to favor optimum formation of the desired enzyme and preferablyis present in a concentration of at least 0.01% (w./v.) of the nutrientmedium.

After a suitable growth period (at least 24 hours), the cells areseparated from the nutrient medium in the usual manner, such as byfiltration or centrifugation, and the separated cells containing thedesired l-dehydrogenase are then mixed with water, an iodoacetate andthe steroid to be l-dehydrogenated, under aerobic conditions.

Among the steroids of the 3,20-diketo-A -pregnene series which may beconverted into useful l-dehydro derivatives by the practice of thisinvention may be mentioned monohydroxyprogesterones (e.g.,Ila-hydroxyprogesterone, the 9aand lla-halo-llp-hydroxyprogesterones,desoxycorticosterone and 2l-fiuoro-17ahydroxyprogesterone); thedihydroxyprogesterones (e.g., corticosterone, the 9aand12a-halocorticosterones, Reichsteinss Compound S,11p,l7a-dihydroxyprogesterone, cortisone, the 91xand 12a-halocortisones,21-fluoro- 1 l5,17adihydroxyprogesterone and 9a,21-difiuoro-11 8,l7a-dihydroxyprogesterone); the trihydroxyprogesterones (e.g.,hydrocortisone, A -pregnene-llu,17a,21-triol-3,20- dione, the and12u-halohydrocortisones, and the 16- methy1-9u-halohydrocortisones); andthe tetrahydroxyprogesterones (e.g.,9cz-fluoro-16a-hydroxyhydrocortisone,6-methyl-9e-fluoro-16a-hydroxyhydrocortisone and the6,9-dihalo-16a-hydroXyhyd-rocortisones); as well as the Zl-esterderivatives of those steroids containing a 21- hydroxyl group (e.g.,Compound S acetate, hydrocortisone acetate, 9u4fluorohydrocortisoneacetate and 9a-fiuorocortisone acetate). The preferred 21-esters arethose of hydrocarbon carboxylic acids having less than ten carbon atomsas exemplified by the lower fatty, acids (e.g., acetic and propionicacids), the monocyclic aryl carboxylic acids (e.g., benzoic and a-toluicacids), the monocyclic aryl lower alkanoic acids (e.g., phenacetic andfi-phenylpropionic acids), the cycloalkane carboxylic acids and thecycloalkene carboxylic acids.

Although the process of this invention is being described as oneemploying separated cells, the process can also be carried out usingeither isolated l-dehydrogenase enzyme or the entire nutrient mediumcontaining the desired microorganism. However, because of the extra costinvolved in isolating the former and the difficulty of inhibitingundesired enzyme action in the latter, the preferred method is, ashereinbefore stated, one where the desired microorganism is firstcultured and the separated cells containing l-dehydrogenase enzyme arethen used to efiect l-dehydrogenation.

To carry out the process of this invention the separated cells aresuspended in an aqueous medium and 3 the iodoacetate compound andsteroid to be converted are added tothe medium, the steroid preferablybeing added either after or with the iodoacetate compound, and theiodoacetate compound being added in a preferred concentration of about0.001 molar to about 0.1 molar and optimally about 0.005 molar to about0.02 molar. An adequate supply of oxygen is also provided, prefer- Iably either by aerating or'agitating the mixture, or both.

following medium (A) contained in three 250 ml. Erlen- Percent Glucose 3Peptone 1 Yeast extract 0.25 KH PO 0.1 Ucon antifoam (a polyalkyleneglycol) 0.01

in distilled water, pH adjusted to 7.0 before autoclaving at 121 forminutes.

The inoculated flasks are incubated at 25 with rotary shaking at 280cycles per minute in a radius of about one'inch. After 48 hours, eachflask is emptied into of chloroform. The tank medium is agitated by animpeller running at 220 r.p.m. and is aerated at the rate of 2.3 cubicfeet per minute. vAfter 48 hours incubation, the cells are harvested bycentrifugation and the cell pastc'obtained is frozen. These cells have1- dehydrogenase activity; g V

(b) Steroid dehydrogenazi0n.A mixture of l g. of the cell paste (wetweight) obtained in step (a), 10 mmoles'of Na HPO 0.8 mmole of sodiumiodoacetate and sufficient water to make 30 ml; in a 250 ml. Erlenmeyer' flask is adjusted to pH 7.8 with N sulfuric acid and incubated at30 for 20 minutes. fiuoro-l6a-hydroxyhydrocortisone, dissolved in 0.176ml. of dimethyl formamide plus 20 ml. of water is then added. Theresulting mixture .is incubated at 30 for two hours while shaking at 250c.p.m. on a rotary shaker with a three-quarter inch stroke. 5 ml. of theresulting mixture is removed, treated with 0.2 ml. of 10 N sulfuric acidand extracted with 2 ml. of methyl isobutyl ketone. The extract is thenanalyzed for steroid con- 10.8 mg. of 9.1-

A drocortisone.

enzyme is prepared by culturing a l-dehydrogenating'fluoro-l6u-hydroxyhydrocortisone.

tent by paper chromatography which reveals a 77% yield of triamcinolone.

'By way of contrast, if the sodium iodoacetate is omitted in step (b) ofExample 1, the yield of triamcinolone is decreased from 77% to 68% andthe yield of total triamcinolone and starting steroid (i.e.,9a-fluoro-l6a-hydroxyhydrocortisone) recovered decreased from 97% to73%.

EXAMPLE 2 Following the procedure of Example 1, step (b) but adjustingthe initial mixture to pH 8.8, the temperature to 35 and the sodiumiodoacetate to 0.25 mmole in 50 ml. of final volume, triamicinolone isobtained in a 78% yield.

If the sodium iodoacetate is omitted in Example 2, the yield oftriamcinolone is'decreased from 78% to 62% and the yield of totaltriamcinolone and starting steroid recovered from 99% to 67%.. r

EXAMPLE 3 Prednz'solone Following the procedure of Example 1, step (b)but substituting 10 mg. of'hydrocortisone for the 9a-fiuoro-16a-hydroxyhydrocortisone, prednisolone is obtained.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

What is claimed is:

1. A process for preparing a. l-dehydro steroid which comprisessubjecting under aerobic conditions a steroid of the 3,20-diketo-A-pregnene series to the action of a l-dehydrogenase enzyme in thepresence of an iodoacetate compound selected from the group consistingof iodoacetic acid, salts thereof and esters thereof, said iodoacetatecompound being present in a molar concentration of at least about 0.001.

2. The process of claim 1 wherein the iodoacetate compound is an alkalimetal iodoacetate.

3. The process of claim 1 wherein the iodoacetate compound is sodiumiodoacetate.

4. The process of claim 1 wherein the steroid is 5. The process of claim1 wherein the steroid is hy- 6. The process of'claim l wherein thel-dehydrogenase strain of a microorganism in the presence of a1,2-saturated steroid." I

References Cited in the file of this patent UNITED STATES PATENTSWettstein et a1. July 22, 1958 Charney Aug. 30, 1960 OTHER REFERENCESBergeys Manual, 7thedition, The Williams and Wilkins Co., Baltimore, Md,1957, page 1018', POSL,

1. A PROCESS FOR PREPARING A 1-DEHYDRO STEROID WHICH COMPRISESSUBJECTING UNDER AEROBIC CONDITIONS A STEROID OF THE3,20-DIKETO-$4-PREGNENE SERIES TO THE ACTION OF A 1-DEHYDROGENASE ENZYMEIN THE PRESENCE OF AN IODOACETATE COMPOUND SELECTED FROM THE GROUPCONSISTING OF IODOACETIC ACID, SALTS THEREOF AND ESTERS THEREOF, SAIDIODOACETATE COMPOUND BEING PRESENT IN A MOLAR CONCENTRATION OF AT LEASTABOUT 0.001.